The present invention relates to an adjustable distribution cell apparatus for chromatogrphic columns, and more particularly to such an apparatus having twin sealing mechanisms adapted to seal a small volume of eluant (mobile phase medium) at working pressure therebetween. This invention also relates to chromatography columns useful with the inventive distribution cell apparatus.
The use of column chromatography to separate chemical compounds is well known. The separation of chemical species occurs due to interaction between the sample, the stationary phase and the mobile phase. The stationary phase is a dispersed medium, with a large surface area, through which the mobile phase is allowed to flow. The chemical nature of the stationary phase exercises the primary control over the separation process; the greater the affinity of a particular chemical compound (the solute) for the stationary medium, the longer it will be retained in the system. Various attraction forces are responsible for retention, including Van de Waals interactions, hydrogen bonding, ionic bonding and reversible complex formation such as the antigen-antibody reaction.
Chromatographic columns are comprised of relatively few parts. A cylindrical column filled with a stationary phase medium is bounded by a bottom cell which permits the mobile phase eluant but not the stationary phase to pass therethrough. A top or "distribution" cell applies the sample and mobile phase across the top of the stationary medium. In many columns the bottom cell is identical to the distribution cell, (in which case both are called "end cells") a configuration which permits the column be run in either direction. Seals must be provided to ensure that the eluant and sample do not leak beyond the end cells and out of the column.
During column chromatography, the mobile phase is percolated through the column by means of gravity or under pressure. High pressure stainless steel industrial columns may operate at up to 3000 p.s.i. (200 kg/cm.sup.2), while acrylic, glass and standard stainless steel columns are commonly operated in the range of one atmosphere (approx. 15 p.s.i.) to 4 atmospheres, 3 atmospheres and 6.5 atmospheres, respectively. The choice of stationary phase and mobile phase media of course is critical, but will not be discussed here. Silicas are commonly chosen as stationary phase, although separation of biomolecules, based on size rather than charge, is often accomplished with porous gels. Commonly employed mobile media include the non-polar straight chain hydrocarbons, aromatic hydrocarbons and ethers and ketones of intermediate polarity and the highly polar alcohols and water.
Although column chromatography in the laboratory has long been practiced in glass biurets containing hand-packed stationary phase, the adaptation of column chromatography to large scale industrial separation processes has required the "fine tuning" of the chromatographic technique. For example, it is known that in order to produce accurate separation of the sample it is necessary to distribute the sample uniformly across the top of the column of stationary medium. Moreover, the sample should be distributed directly onto the medium, i.e., with a minimum of "head space" between the top of the medium and the distribution cell which applies the sample thereto. Because the height of the packed column may vary from use to use depending on the chemicals to be separated, or due to swelling or settling of the stationary medium immediately after packing, adjustable distribution cells have won acceptance in history.
U.S. Pat. No. 3,487,938 relates to an "automatic head space reducer" and provides a movable piston which rests on the column of chromatographic medium. The piston automatically follows the top of the column to minimize head space should the bed swell or contract.
U.S. Pat. No. 3,483,986 (A. G. Wright) relates to column chromatography apparatus. Disclosed therein are adjustable end cell apparatus useful for distributing eluant and sample across the top of the column bed. Compressible o-rings form a fluid tight seal around the cells.
Published European Patent Office Patent Application No. 8,921 (A. G. Wright) also discloses adjustable end cell apparatus for chromatographic columns.
As previously mentioned, columns may be manufactured from acrylic, glass or stainless steel. The height adjustment of the distribution cell is easily made in a clear acrylic or glass column as both the top of the column bed and the bottom of the distribution cell are visible. Adjustment of the distribution cell in a stainless steel column is difficult because neither the distribution cell nor the top of the packed bed are visible within the column. To remedy this disadvantage in otherwise desirable stainless steel columns, one aspect of this invention provides a stainless steel column with a plurality of viewing windows disposed therein. These windows are positioned in order to enable the column operator to view the top of the packed bed and to properly position the distribution cell at the top of the bed.
Another aspect of this invention provides an improved adjustable distribution cell apparatus for use with chromatography columns, especially a column with windows. The inventive apparatus provides a seal adjust tube which carries independently operable first and second sealing elements for forming fluid tight seals between the distribution cell and the column tube. A band of eluant, preferably at the working pressure of the column, is allowed to form between the first and second seals and ensures a tight seal in the window region of the column.